Many important cellular and physiological events, including nutrient uptake, signal transduction and cell cycle progression are mediated by transmembrane ion gradients. An extensive, multigene family of cation pumps, the P-ATPases, have evolved to transport a wide variety of different ions (Ca2+, Na+, K+, H+, Mg2+, Cu2+, to name a few). In keeping with their essential roles, the P-ATPases are a target for pharmacological intervention in disease (such as congestive heart failure and stomach ulcers), and are defective in various inherited disorders (Menkes, Wilson, Brody and Hailey-Hailey disease). Despite the similarities in sequence, structure and mechanism within this family, individual members differ strikingly in ion selectivity. The molecular basis of selectivity in ion pumps remains one of the fundamental unanswered problems in the field of membrane bioenergetics. To approach this problem, we will: focus on the Golgi Ca2+, Mn2+-ATPase, Pmrl, in the genetically tractable organism yeast, apply simple and powerful phenotypic screens that will identify loss of function or selectivity mutations, develop rigorous biochemical tools to analyze the defective pumps. In Aim 1, we will identify the molecular determinants of divalent cation selectivity in yeast Pmrl, a founding member of the newly- defined subgroup of Golgi/secretory pathway Ca2+-ATPases. Specifically, we will focus on selectivity for Mn2+ versus Ca2+ ions. In one approach, we will use directed and random mutagenesis techniques in conjunction with biological assays for Ca2+ chelator and Mn2+ toxicity to identify mutations that alter ion selectivity. In a second approach, we will use homology modeling of yeast Pmrl, based on the known crystal structure of the SERCA pump, to design rational targets for mutagenesis. Target residues will include those predicted to line the ion conducting pathway, stabilize adjacent membrane helices or form domain interfaces. In Aim 2, loss-of-function mutants with interesting properties such as alterations in ion selectivity or uncoupling of ATPase hydrolysis from ion transport, will be further mutagenized and subjected to phenotypic selection in order to identify intragenic suppressor mutations. These will provide unique insight on critical interactions between domains, and within or between membrane helices, that will complement structural information on ion pumps. In Aim 3, large-scale purification of Pmrl from fermentor-grown Pichia pastoris cultures will be undertaken for structural studies on cation binding and the concomitant conformational changes. Taken together, these aims constitute a powerful approach toward deciphering the molecular basis of selectivity and transport in ion pumps.